Nitrated-fatty acids modulation of type II diabetes

ABSTRACT

Nitro oleic acid and related metabolites are agonists of PPAR-γ. Surprisingly, nitro oleic acid is a more potent agonist of PPAR-γ, relative to nitro linoleic acid. Thus, nitro oleic acid and its metabolites, as well as their pharmaceutically acceptable salts and prodrug forms, are candidate therapeutics for the treatment of type-2 diabetes, which results from insulin resistance accompanying the improper functioning of PPAR-γ.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This is a Continuation of U.S. patent application Ser. No. 14/923,265,filed Oct. 26, 2015, issued as U.S. Pat. No. 9,700,534 Jul. 11, 2017,which is a Continuation of U.S. patent application Ser. No. 14/244,741,filed Apr. 3, 2014, issued as U.S. Pat. No. 9,186,408 Nov. 17, 2015,which is a Continuation of U.S. patent application Ser. No. 13/666,827,filed Nov. 1, 2012, issued as U.S. Pat. No. 8,735,449 May 27, 2014,which is a continuation of U.S. patent application Ser. No. 12/670,951,filed May 10, 2010, issued Dec. 4, 2012, as U.S. Pat. No. 8,324,277,which is the U.S. National Stage of International Application No.PCT/US2008/09274, filed Aug. 1, 2008, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 60/953,360, filed Aug. 1, 2007, all of whichare hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with United States government support undergrant number R01 HL058115, awarded by the National Institutes of Health.The United States government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates to the use of nitrated fatty acids astherapeutics for treating type-2 diabetes. Fatty acids are bothphysiological energy sources and mediators of signaling events involved,for example, in inflammation and in energy homeostasis.

Saturated, unsaturated, and polyunsaturated fatty acids have beenidentified to date. Unsaturated electrophilic fatty acids have emergedas an important class of endogenous signaling molecules. Within thisclass are fatty acid hydroperoxides, keto fatty acids, and nitratedfatty acids, among others. For example, see Freeman et al., Chem. Res.Toxicol. 12: 83-92 (1999), and Lima et al., Biochemistry 41: 10717-22(2002).

The signaling ability of nitro fatty acids stems predominantly fromtheir ability to form reversible covalent adducts with nucleophiliccenters of cellular proteins that are implicated in varioustranscriptional and cellular signaling processes. In particular,regulation of signaling activity most often occurs via the covalentmodification of an active site thiol group of a protein target.

Recent studies suggest that nitro fatty acids such as 9- or 10-nitrooctadecenoic acid (“nitro oleic acid”) and the various regioisomers (9-,10-, 12- and 13-nitro) of nitro linoleic acid are adaptive mediatorsthat play a crucial role in linking disease processes with underlyingcellular events. See Freeman et al., Proc. Nat'l Acad. Sci. USA 99:15941-46 (2002). In particular, nitro fatty acids modulate the activityof the peroxisome proliferator activating receptor gamma (PPAR-γ), forexample, in response to inflammation and metabolic imbalance.

While both nitro oleic acid and nitro linoleic acid interact withPPAR-γ, little is known about the structural and biochemicaldeterminants that account for their PPAR-γ activity and the relateddownstream activation of gene transcription. Consequently, no systematicapproach exits for the design of pharmacophores that can modulate PPAR-γactivity.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides, in one of its aspects, apharmaceutical composition comprising (A) an active agent selected fromnitro oleic acid and a metabolite of nitro oleic acid, or apharmaceutically acceptable salt or prodrug of such active agent, and(B) a pharmaceutically acceptable carrier. In a preferred embodiment,the active agent is nitro oleic acid.

In accordance with another of its aspects, the invention provides amethod for treating type-2 diabetes, comprising (A) administering to asubject in need thereof a pharmaceutical composition as described aboveand then (B) repeating step (A) at least once. Preferably, the methodfurther comprises, after at least one repetition of step (A); themonitoring of the subject for a change relating to type-2 diabetes.

Pursuant to yet another aspect of the invention, a method is providedfor gauging efficacy of a treatment for type-2 diabetes. This methodcomprises (A) obtaining a first and a second sample from a subjectsuffering from type-2 diabetes, which samples are obtained at differenttimes during said treatment; (B) determining blood glucose levels in thefirst and second samples; and (C) comparing the blood glucose levelbetween the samples. In accordance with these steps, a lower bloodglucose level in the second sample is an indicator of efficacy of thetreatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting serum levels of nitro oleic acid and as itsphysiological metabolites as a function of time.

FIG. 2 is a graph that correlates blood glucose levels in wild-type (WT)mice on different days after injection with oleic acid, nitro oleicacid, rosiglitazone, and a vehicle.

FIG. 3 is a graph that correlates blood glucose levels inleptin-deficient diabetic ob/ob mice on different days after beinginjected with oleic acid, nitro oleic acid, rosiglitazone, and avehicle.

FIG. 4 is a graph that correlates the body weights of WT and ob/ob mice,undergoing treatment with nitro oleic acid, to the body weight of WT andob/ob mice that receive oleic acid, rosiglitazone, and a vehicle,respectively.

FIG. 5 is a graph that shows the change in insulin sensitivity for WTmice injected with oleic acid, nitro oleic acid, rosiglitazone, and avehicle, respectively

FIG. 6 is a graph that documents a change in insulin sensitivity ofob/ob mice, observed after injection with oleic acid, nitro oleic acid,rosiglitazone, and a vehicle, respectively.

DETAILED DESCRIPTION OF THE INVENTION

Nitro oleic acid and related metabolic products (“metabolites”) areagonists of PPAR-γ. The inventor's discovery that nitro oleic acid issurprisingly more potent as an agonist of PPAR-γ, relative to nitrolinoleic acid, underscores the prospect, in accordance with the presentinvention, of using nitro oleic acid and its metabolites, as well astheir pharmaceutically acceptable salts and prodrug forms, as activeagents in the treatment of type-2 diabetes, which results from insulinresistance accompanying the improper functioning of PPAR-γ.

The structural determinants responsible for the potency of nitro oleicacid and its metabolites at PPAR-γ were illuminated using acomputational model for receptor-ligand interaction. Modeling dataindicate that both arginine-288 (Arg288) and cysteine-285 (Cys285),present in the ligand binding pocket of PPAR-γ, are important forbinding. For example, the receptor-ligand model indicates anelectrostatic interaction between arginine-288 and the anionic nitrogroup of nitro oleic acid, while Cys285 is found to be in a suitableposition for interacting with the olefinic double bond. Theseobservations indicate that nitro oleic acid activates PPAR-γ via thecovalent modification of its active site thiol, and compounds thatpreserve such interactions will activate the receptor in a similarmanner, thus qualifying as a candidate therapeutic for treating type-2diabetes.

Analysis of mouse plasma after intravenous administration of nitro oleicacid illuminates the physiological fate of nitro oleic acid. Nitro oleicacid is converted in-vivo, to its saturated analog, or can undergoβ-oxidative cleavage to give several short chain products, such as thecorresponding saturated or unsaturated C-10 to C-16 nitrated analogs.FIG. 1 shows the plasma levels of nitro oleic acid or its metabolicproducts as a function of time. For nitro oleic acid as well as itssaturated 18:0 nitrated analog, the curve is biphasic, with peakconcentrations occurring at around 5 minutes following theadministration of nitro oleic acid. In contrast, the plasma levels forthe β-oxidation products are highest at around 60 minutes.

The presence of β-oxidation products in blood plasma has importantphysiological implications. It is believed that the short-chainmetabolites are less hydrophobic than the parent acid. Nevertheless,these compounds preserve the molecular determinants that are believed tobe important for binding. Additionally, the smaller size the C-10 toC-16 metabolic products will allow these metabolites to partitiondifferently between the hydrophobic and hydrophilic compartmentsphysiologically. Such differences in partitioning ratios alter theanatomic distribution, chemical reactivity, and pharmacological profilesof these metabolites, by altering their availability to cellulartargets. Pursuant to the invention, the C-10 to C-16 metabolites arealso suitable candidate therapeutics for the treatment of type-2diabetes, a condition associated with PPAR-γ dysfunction. See Freeman etal., Chem. Res. Toxicol. 12: 83-92 (1999).

Supportive of this anti-diabetic indication, nitro oleic acid was foundto improve insulin sensitivity and lower blood glucose levels in ob/obmice. In particular, in-vivo results indicate that nitro oleic acidreduces blood glucose levels without the side-effects of weight gain andfluid retention associated with Rosiglitazone, a known PPAR-γ agonist.

As shown in FIG. 2, nitro oleic acid but not oleic acid maintains asteady blood glucose level in fed WT mice. The in-vivo results indicatethat nitro oleic acid was at least as effective as Rosiglitazone inmaintaining blood glucose levels. Similar results are observed inexperiments involving ob/ob mice. As seen from the graph in FIG. 3, bothnitro oleic acid and Rosilitazone are effective in reducing bloodglucose levels. However, for mice receiving oleic acid the blood glucoselevels increased over the course of the study. These results, therefore,provide support for nitro oleic acid's role in lowering blood glucoseand as a candidate therapeutic for the treatment of diabetes.

In addition to reducing blood glucose levels, no increase in body weightis observed when nitro oleic acid is administered to mice. As shown inFIG. 4, the body weights of WT mice receiving nitro oleic acid, orRosiglitazone do not change over the course of the study (25 days),however, the results are substantially different for ob/ob mice. In thiscase, the body weight initially decreases for animals receiving nitrooleic acid (days 0-10) and then remains constant over the latter half ofthe study. In contrast, animals receiving Rosilitazone or oleic acidshow a steady increase in body weights over the entire course of thestudy.

Without endorsement of any particular theory, the absence of weight gainin ob/ob mice is believed to occur because physiologically nitro oleicacid is adduced to plasma, which serves as a “storage system” andtemporarily inactivates the nitrated fatty acid, until it is requiredfor facilitating a particular signal transduction event. Sinceactivation of PPAR-γ occurs upon binding free nitro oleic acid, thesequestration of this molecule prevents the aberrant activation ofPPAR-γ or the transcription of genes that are regulated by this nuclearreceptor.

Further evidence, that nitro oleic acid modulates PPAR-γ activity via abinding interaction different from that of Rosiglitazone is provided bythe discovery that nitro oleic acid and not Rosiglitazone improvesinsulin sensitivity in ob/ob mice. In this regard, for WT mice receivingeither nitro oleic acid or Rosiglitazone, the administration of insulinresults in an initial drop in blood glucose levels followed by anelevation to normal levels shortly after the administration of insulinas shown in FIG. 5. In contrast, as seen by the graph in FIG. 6,administration of nitro oleic acid to ob/ob mice followed by theadministration of insulin causes a substantial decrease in blood glucoselevels. On the other hand, the blood glucose levels in ob/ob micereceiving Rosiglitazone are unchanged upon administration of insulin.These results indicate that administration of nitro oleic prior toinsulin enhances insulin sensitivity in ob/ob diabetic mice, whileRosiglitazone fails to do so. The fact that both nitro oleic acid andRosiglitazone exert their blood glucose lowering effect through theactivation of PPAR-γ, indicates that they interact differently with thereceptor and consequently the transcription of genes that regulatemetabolic events that lead to weight gain and fluid retention.

The importance of nitro fatty acids as signaling molecules has promptedthe development of various procedures for synthesizing these compounds.For example, Brandchaud et al., Org. Lett. 8: 3931-34 (2006), and Kinget al., Org. Lett. 8: 2305-08 (2006), disclose syntheses that could beused in the context of the present invention. Another suitable synthesisapproach, disclosed in U.S. Patent Publication No. 2007/0232579,involves the direct nitration of an appropriate unsaturated fatty acid.Accordingly, (Z)-octadec-9-enoic acid (oleic acid) is reacted with NaNO₂in the presence of phenylselenium bromide and mercuric chloride underanhydrous conditions to give 9-nitro or 10-nitro oleic acid. A similarsynthetic strategy is believed to nitrate the appropriate C-10 to C-16unsaturated fatty acids to give the corresponding nitro oleic acidmetabolic products.

Thus obtained, the synthetic regioisomers of nitro oleic acid or theirrespective C-10 to C-16 metabolites are typically purified prior tobiological use. In one aspect of the invention, therefore, large-scalepurification of the individual isomers is carried out using preparativehigh performance liquid chromatography (HPLC), as described in U.S.Patent Publication No. 2007/0232579. The purified compounds thusobtained are appropriately formulated prior to in vivo administration.

A pharmaceutical composition of the invention can include one or moretherapeutic agents in addition to nitro oleic acid or a relatedcompound, as described above. Illustrative of such therapeutics arecytokines, chemokines, and/or regulators of growth factors.Additionally, the invention contemplates a formulation that containseither a single regioisomer or both regioisomers of nitro oleic acid.

The pharmaceutical composition can have more than one physiologicallyacceptable carrier, too, such as a mixture of two or more carriers. Thecomposition also can include thickeners, diluents, solvents, buffers,preservatives, surface active agents, excipients, and the like.

The pharmaceutical carrier used to formulate the nitro oleic acid of theinvention will depend on the route of administration. Administration maybe topical (including opthamalic, vaginal, rectal, or intranasal), oral,by inhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection.

Thus, nitro oleic acid or its metabolites can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, transdermally, intratracheally, extracorporeally, ortopically (e.g., topical intranasal administration or administration byinhalant). In this regard, the phrase “topical intranasaladministration” connotes delivery of the compositions into the nose andnasal passages through one or both of the nares and can comprisedelivery by a spraying mechanism or droplet mechanism, or throughaerosolization of the nucleic acid or vector. The latter can beeffective when a large number of subjects are to be treatedsimultaneously, where “subject” can denote a human or an non-humananimal. Administration of the compositions by inhalant can be throughthe nose or mouth via delivery by a spray or droplet mechanism. Deliveryalso can be directed to any area of the respiratory system, such as thelungs, via intubation.

A pharmaceutical composition of the nitro oleic acid and its metabolitesfor parenteral administration, according to the invention, can includeexcipients and carriers that stabilize the nitro fatty acid mimetic.Illustrative of such a carrier are non-aqueous solvents, such aspropylene glycol, polyethylene glycol, vegetable oils, and injectableorganic esters such as ethyl oleate. Additionally, formulations forparenteral administration include liquid solutions, suspensions, orsolid forms suitable for solution or suspension in liquid prior toinjection, or emulsions.

Intravenous compositions can include agents to maintain the osmomolarityof the formulation. Examples of such agents include sodium chloridesolution, Ringer's dextrose, dextrose, lactated Ringer's solution, fluidand nutrient replenishers, and the like. Also included in intravenousformulations are one or more additional ingredients that preventmicrobial infection or inflammation, as well as anesthetics.

Alternatively, pharmaceutical compositions of the salt form of nitrooleic acid or its metabolites is administered. Illustrative of suchsalts are those formed by reaction of the carboxyl group with aninorganic base such as sodium hydroxide, ammonium hydroxide, orpotassium hydroxide. Additionally, the salt is formed by reacting thecarboxyl group with organic bases such as mono-, di-, trialkyl and arylamines and substituted ethanolamines.

To address concerns that at physiological pH, nitro oleic acid typicallywill be a negatively charged molecule, which may have non-optimalbioavailability and cell-transport kinetics, one may provide a compoundof the invention formulated as a prodrug. Illustrative of such a prodrugis a pharmaceutically acceptable ester, such as a methyl or an ethylester. The ester acts as a prodrug because non-specific intracellularesterase convert it to the active form responsible for elicitingtherapeutic effect.

Type-2 diabetes is a chronic condition that results from a loss ofsensitivity to insulin. As described above, a pharmaceutical compositionof the invention improves insulin sensitivity and, hence, can serve as atherapeutic for treating type-2 diabetes. Successful treatment of type-2diabetes typically entails as well an ongoing monitoring of the subjectfor changes related to the diabetic condition, e.g., monitoringphysiological levels of different metabolic parameters associated withthis condition. Thus, the subject's blood and urine glucose levels canbe measured to assess how frequently to administer the inventivecomposition. Additional markers such as a gain in body weight, frequencyof urination and the levels of glucagon in the blood can be used tomonitor and possibly to modify treatment to best suit the given subject.

In support of such an anti-diabetic regimen, the present invention alsoprovides for using one of the above-mentioned active agents to prepare apharmaceutical composition for treating type-2 diabetes in a subject. Tothis end, different formulation approaches have been described above.

In a related vein, the invention encompasses a method for gauging thetherapeutic efficacy of the composition as described above. This methodinvolves obtaining at least two blood samples from a subject atdifferent times during treatment and measuring the level of bloodglucose in each sample. Indicative of therapeutic efficacy is a lowerlevel of blood glucose in the sample obtained at a later time pointduring treatment. As shown in FIG. 3, blood glucose levels in ob/ob micereceiving nitro oleic acid are significantly lower on day 21 than at thebeginning of the study, indicating the therapeutic benefit of nitrooleic acid in treatment of type 2 diabetes.

What is claimed is:
 1. An oral unit dosage form comprising apharmaceutical composition, the pharmaceutical composition comprising(A) an active agent selected from nitro oleic acid and a metabolite ofnitro oleic acid, or a pharmaceutically acceptable salt or prodrug ofsaid active agent, and (B) a pharmaceutically acceptable carrier.
 2. Theoral unit dosage form of claim 1, wherein the active agent is nitrooleic acid.